title: Enzymatic properties of Populus α- and β-NAD-ME recombinant proteins.
abstract: Plant mitochondrial NAD-malic enzyme (NAD-ME), which is composed of α- and β-subunits in many species, participates in many plant biosynthetic pathways and in plant respiratory metabolism. However, little is known about the properties of woody plant NAD-MEs. In this study, we analyzed four NAD-ME genes (PtNAD-ME1 through PtNAD-ME4) in the genome of Populus trichocarpa. PtNAD-ME1 and -2 encode putative α-subunits, while PtNAD-ME3 and -4 encode putative β-subunits. The Populus NAD-MEs were expressed in Escherichia coli cells as GST-tagged fusion proteins. Each recombinant GST-PtNAD-ME protein was purified to near homogeneity by glutathione-Sepharose 4B affinity chromatography. Milligram quantities of each native protein were obtained from 1 L bacterial cultures after cleavage of the GST tag. Analysis of the enzymatic properties of these proteins in vitro indicated that α-NAD-MEs are more active than β-NAD-MEs and that α- and β-NAD-MEs presented different kinetic properties (Vmax, kcat and kcat/Km). The effect of different amounts of metabolites on the activities of Populus α- and β-NAD-MEs was assessed in vitro. While none of the metabolites evaluated in our assays activated Populus NAD-ME, oxalacetate and citrate inhibited all α- and β-NAD-MEs and glucose-6-P and fructose inhibited only the α-NAD-MEs.
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China. chengyuxiang@nefu.edu.cn.
authors.forename: Jinwen
authors.initials: J
authors.lastname: Liu
authors.forename: Qiguo
authors.initials: Q
authors.lastname: Yu
authors.forename: Nabil I
authors.initials: NI
authors.lastname: Elsheery
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Cheng
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D032107:Populus / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D009243:NAD